Cyclic AMP-specific phosphodiesterase inhibitors

ABSTRACT

Novel compounds that are potent and selective inhibitors of PDE4, as well as methods of making the same, are disclosed. Use of the compounds in the treatment of inflammatory diseases and other diseases involving elevated levels of cytokines, as well as central nervous system (CNS) disorders, also is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of provisional application Ser.No. 60/171,950, filed Dec. 23, 1999.

FIELD OF INVENTION

[0002] The present invention relates to a series of compounds that arepotent and selective inhibitors of cyclic adenosine 3′,5′-monophosphatespecific phosphodiesterase (cAMP specific PDE). In particular, thepresent invention relates to a series of novel thiazole compounds thatare useful for inhibiting the function of cAMP specific PDE, inparticular, PDE4, as well as methods of making the same, pharmaceuticalcompositions containing the same, and their use as therapeutic agents,for example, in treating inflammatory diseases and other diseasesinvolving elevated levels of cytokines and proinflammatory mediators.

BACKGROUND OF THE INVENTION

[0003] Chronic inflammation is a multi-factorial disease complicationcharacterized by activation of multiple types of inflammatory cells,particularly cells of lymphoid lineage (including T lymphocytes) andmyeloid lineage (including granulocytes, macrophages, and monocytes).Proinflammatory mediators, including cytokines, such as tumor necrosisfactor (TNF) and interleukin-1 (IL-1), are produced by these activatedcells. Accordingly, an agent that suppresses the activation of thesecells, or their production of proinflammatory cytokines, would be usefulin the therapeutic treatment of inflammatory diseases and other diseasesinvolving elevated levels of cytokines.

[0004] Cyclic adenosine monophosphate (cAMP) is a second messenger thatmediates the biologic responses of cells to a wide range ofextracellular stimuli. When the appropriate agonist binds to specificcell surface receptors, adenylate cyclase is activated to convertadenosine triphosphate (ATP) to cAMP. It is theorized that the agonistinduced actions of cAMP within the cell are mediated predominately bythe action of cAMP-dependent protein kinases. The intracellular actionsof cAMP are terminated by either a transport of the nucleotide to theoutside of the cell, or by enzymatic cleavage by cyclic nucleotidephosphodiesterases (PDEs), which hydrolyze the 3¹-phosphodiester bond toform 5′-adenosine monophosphate (5′-AMP). 5′-AMP is an inactivemetabolite. The structures of cAMP and 5′-AMP are illustrated below.

[0005] Elevated levels of cAMP in human myeloid and lymphoid lineagecells are associated with the suppression of cell activation. Theintracellular enzyme family of PDEs, therefore, regulates the level ofcAMP in cells. PDE4 is a predominant PDE isotype in these cells, and isa major contributor to cAMP degradation. Accordingly, the inhibition ofPDE function would prevent the conversion of cAMP to the inactivemetabolite 5′-AMP and, consequently, maintain higher cAMP levels, and,accordingly, suppress cell activation (see Beavo et al., “CyclicNucleotide Phosphodiesterases: Structure, Regulation and Drug Action,.”Wiley and Sons, Chichester, pp. 3-14, (1990)); Torphy et al., Drug Newsand Perspectives, 6, pp. 203-214 (1993); Giembycz et al., Clin. Exp.Allergy, 22, pp. 337-344 (1992)).

[0006] In particular, PDE4 inhibitors, such as rolipram, have been shownto inhibit production of TNFα and partially inhibit IL-1β release bymonocytes (see Semmler et al., Int. J. Immunopharmacol., 15, pp.409-413, (1993); Molnar-Kimber et al., Mediators of Inflammation, 1, pp.411-417, (1992)). PDE4 inhibitors also have been shown to inhibit theproduction of superoxide radicals from human polymorphonuclearleukocytes (see Verghese et al., J. Mol. Cell. Cardiol., 21 (Suppl. 2),S61 (1989); Nielson et al., J. Allergy Immunol., 86, pp. 801-808,(1990)); to inhibit the release of vasoactive amines and prostanoidsfrom human basophils (see Peachell et al., J. Immunol., 148, pp.2503-2510, (1992)); to inhibit respiratory bursts in eosinophils (seeDent et al., J. Pharmacol., 103, pp. 1339-1346, (1991)); and to inhibitthe activation of human T-lymphocytes (see Robicsek et al., Biochem.Pharmacol., 42, pp. 869-877, (1991)).

[0007] Inflammatory cell activation and excessive or unregulatedcytokine (e.g., TNFα and IL-1β) production are implicated in allergic,autoimmune, and inflammatory diseases and disorders, such as rheumatoidarthritis, osteoarthritis, gouty arthritis, spondylitis, thyroidassociated ophthalmopathy, Behcet's disease, sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shocksyndrome, asthma, chronic bronchitis, adult respiratory distresssyndrome, chronic pulmonary inflammatory disease, such as chronicobstructive pulmonary disease, silicosis, pulmonary sarcoidosis,reperfusion injury of the myocardium, brain, and extremities, fibrosis,cystic fibrosis, keloid formation, scar formation, atherosclerosis,transplant rejection disorders, such as graft vs. host reaction andallograft rejection, chronic glomerulonephritis, lupus, inflammatorybowel disease, such as Crohn's disease and ulcerative colitis,proliferative lymphocyte diseases, such as leukemia, and inflammatorydermatoses, such as atopic dermatitis, psoriasis, and urticaria.

[0008] Other conditions characterized by elevated cytokine levelsinclude brain injury due to moderate trauma (see Dhillon et al., J.Neurotrauma, 12, pp. 1035-1043 (1995); Suttorp et al., J. Clin. Invest.,91, pp. 1421-1428 (1993)), cardiomyopathies, such as congestive heartfailure (see Bristow et al., Circulation, 97, pp. 1340-1341 (1998)),cachexia, cachexia secondary to infection or malignancy, cachexiasecondary to acquired immune deficiency syndrome (AIDS), ARC (AIDSrelated complex), fever myalgias due to infection, cerebral malaria,osteoporosis and bone resorption diseases, keloid formation, scar tissueformation, and pyrexia.

[0009] In particular, TNFα has been identified as having a role withrespect to human acquired immune deficiency syndrome (AIDS). AIDSresults from the infection of T-lymphocytes with Human ImmunodeficiencyVirus (HIV). Although HIV also infects and is maintained in myeloidlineage cells, TNF has been shown to upregulate HIV infection inT-lymphocytic and monocytic cells (see Poli et al., Proc. Natl. Acad.Sci. USA, 87, pp. 782-785, (1990)).

[0010] Several properties of TNFα, such as stimulation of collagenases,stimulation of angiogenesis in vivo, stimulation of bone resorption, andan ability to increase the adherence of tumor cells to endothelium, areconsistent with a role for TNF in the development and metastatic spreadof cancer in the host. TNFα recently has been directly implicated in thepromotion of growth and metastasis of tumor cells (see Orosz et al., J.Exp. Med., 177, pp. 1391-1398, (1993)) PDE4 has a wide tissuedistribution. There are at least four genes for PDE4 of which multipletranscripts from any given gene can yield several different proteinsthat share identical catalytic sites. The amino acid identity betweenthe four possible catalytic sites is greater than 85%. Their sharedsensitivity to inhibitors and their kinetic similarity reflect thefunctional aspect of this level of amino acid identity. It is theorizedthat the role of these alternatively expressed PDE4 proteins allows amechanism by which a cell can differentially localize these enzymesintracellularly and/or regulate the catalytic efficiency via posttranslational modification. Any given cell type that expresses the PDE4enzyme typically expresses more than one of the four possible genesencoding these proteins.

[0011] Investigators have shown considerable interest in the use of PDE4inhibitors as anti-inflammatory agents. Early evidence indicates thatPDE4 inhibition has beneficial effects on a variety of inflammatorycells such as monocytes, macrophages, T-cells of the Th-1 lineage, andgranulocytes. The synthesis and/or release of many proinflammatorymediators, such as cytokines, lipid mediators, superoxide, and biogenicamines, such as histamine, have been attenuated in these cells by theaction of PDE4 inhibitors. The PDE4 inhibitors also affect othercellular functions including T-cell proliferation, granulocytetransmigration in response to chemotoxic substances, and integrity ofendothelial cell junctions within the vasculature.

[0012] The design, synthesis, and screening of various PDE4 inhibitorshave been reported. Methylxanthines, such as caffeine and theophylline,were the first PDE inhibitors discovered, but these compounds arenonselective with respect to which PDE is inhibited. The drug rolipram,an antidepressant agent, was one of the first reported specific PDE4inhibitors. Rolipram, having the following structural formula, has areported 50% Inhibitory Concentration (IC₅₀) of about 200 nM (nanomolar)with respect to inhibiting recombinant human PDE4.

[0013] Investigators have continued to search for PDE4 inhibitors thatare more selective with respect to inhibiting PDE4, that have a lowerIC₅₀ than rolipram, and that avoid the undesirable central nervoussystem (CNS) side effects, such as retching, vomiting, and sedation,associated with the administration of rolipram. One class of compoundsis disclosed in Feldman et al. U.S. Pat. No. 5,665,754. The compoundsdisclosed therein are substituted pyrrolidines having a structuresimilar to rolipram. One particular compound, having structural formula(I), has an IC₅₀ with respect to human recombinant PDE4 of about 2 nM.Inasmuch as a favorable separation of emetic side effect from efficacywas observed, these compounds did not exhibit a reduction in undesirableCNS effects.

[0014] In addition, several companies are now undertaking clinicaltrials of other PDE4 inhibitors. However, problems relating to efficacyand adverse side effects, such as emesis and central nervous systemdisturbances, remain unsolved.

[0015] Accordingly, compounds that selectively inhibit PDE4, and thatreduce or eliminate the adverse CNS side effects associated with priorPDE4 inhibitors, would be useful in the treatment of allergic andinflammatory diseases, and other diseases associated with excessive orunregulated production of cytokines, such as TNF. In addition, selectivePDE4 inhibitors would be useful in the treatment of diseases that areassociated with elevated cAMP levels or PDE4 function in a particulartarget tissue.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to potent and selective PDE4inhibitors useful in treatment of diseases and conditions whereinhibition of PDE4 activity is considered beneficial. The present PDE4inhibitors unexpectedly reduce or eliminate the adverse CNS side effectsassociated with prior PDE4 inhibitors.

[0017] In particular, the present invention is directed to compoundshaving the structural formula (II):

[0018] wherein R¹ is lower alkyl, bridged alkyl (e.g., norbornyl),aralkyl (e.g., indanyl), cycloalkyl, a 5- or 6-membered saturatedheterocycle (e.g., 3-tetrahydrofuryl), C₁₋₃alkylenecycloalkyl (e.g.,cyclopentylmethyl), aryl- or heteroaryl-substituted propargyl (e.g.,—CH₂C≡C—CH₅), aryl- or heteroaryl-substituted allyl (e.g.,—CH₂CH═CH—C₆H₅) or halocycloalkyl (e.g., fluorocyclopentyl);

[0019] R² is hydrogen, methyl, or halo-substituted methyl, e.g., CHF₂;

[0020] R³ is selected from the group consisting of C(═O) OR⁷, C(═O) R⁷,C(═NH) NRR⁹, C(═O) NR⁸R⁹, lower alkyl, bridged alkyl, cycloalkyl,haloalkyl, halocycloalkyl, C₁₋₃alkylenecycloalkyl, a 5- or 6-memberedsaturated heterocycle, aryl, heteroaryl, heteroarylSO₂, aralkyl,alkaryl, heteroaralkyl, heteroalkaryl, C₁₋₃alkyleneC(═O)OR⁷,C(═O)C₁₋₃alkyleneC(=O)OR⁷, C₁₋₃alkyleneheteroaryl, C(═O)C(═O)OR⁷,C(═O)C₁₋₃alkyleneC(═O)OR⁷, C(═O)C₁₋₃alkyleneNHC(═O)OR⁷,C(═O)C₃alkyleneNH₂, and NHC(═O) OR⁷;

[0021] R⁴ is hydrogen, lower alkyl, haloalkyl, cycloalkyl, aryl,C(═O)R⁷, OR⁸, NR⁸R⁹, or SR⁸;

[0022] R⁵ is hydrogen, lower alkyl, haloalkyl, cycloalkyl, or aryl;

[0023] R⁷ is selected from the group consisting of cycloalkyl, branchedor unbranched lower alkyl, heteroaryl, a heterocycle, aralkyl, and aryl,and R⁷ is optionally substituted with one or more of OR⁸, NR⁸R⁹, or SR⁸;

[0024] R⁸ and R⁹, same or different, are selected from the groupconsisting of hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl,alkaryl, heteroaralkyl, heteroalkaryl, and aralkyl, or R⁸ and R⁹ aretaken together form a 4-membered to 7-membered ring;

[0025] R¹⁰ is hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, C(═O)alkyl,C(═O)cycloalkyl, C(═O)aryl, C(═O)Oalkyl, C(═O)Ocycloalkyl, C(═O)aryl,CH₂O H, CH₂Oalkyl, CHO, CN, NO₂, or SO₂R¹¹; and

[0026] R is alkyl, cycloalkyl, trifluoromethyl, aryl, aralkyl, or NR⁸R⁹.

[0027] The present invention also is directed to pharmaceuticalcompositions containing one or more of the compounds of structuralformula (II), to use of the compounds and compositions containing thecompounds in the treatment of a disease or disorder, and to methods ofpreparing compounds and intermediates involved in the synthesis of thecompounds of structural formula (II).

[0028] The present invention also is directed to methods of treating amammal having a condition where inhibition of PDE4 provides a benefit,of modulating cAMP levels in a mammal, of reducing TNFα levels in amammal, of suppressing inflammatory cell activation in a mammal, and ofinhibiting PDE4 function in a mammal by administering to the mammal atherapeutically effective amount of a compound of structural formula(II), or a composition containing a composition of structural formula(II).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention is directed to compounds having thestructural formula (II):

[0030] wherein R¹ is lower alkyl, bridged alkyl (e g., norbornyl),aralkyl (e.g., indanyl), cycloalkyl, a 5- or 6-membered saturatedheterocycle (e.g., 3-tetrahydrofuryl), C₁₋₃alkylenecycloalkyl (e.g.,cyclopentylmethyl), aryl- or heteroaryl-substituted propargyl (i.e.,—CH₂C≡C—C₆H⁵), aryl- or heteroaryl-substituted allyl (e.g.,—CH₂CH═CH—C₆H₅) or halocycloalkyl (e.g., fluorocyclopentyl);

[0031] R² is hydrogen, methyl, or halo-substituted methyl, e.g., CHF₂;

[0032] R³ is selected from the group consisting of C(═O) OR⁷, C(═O) R⁷,C(═NH)NR⁶R⁹, C(═O)NR⁸R⁹, lower alkyl, bridged alkyl, cycloalkyl,haloalkyl, halocycloalkyl, C₁₋₃alkylenecycloalkyl, a 5- or 6-memberedsaturated heterocycle, aryl, heteroaryl, heteroarylSO₂, aralkyl,alkaryl, heteroaralkyl, heteroalkaryl, C₁₋₃alkyleneC(═O)OR⁷,C(═O)C₁₃alkyleneC(═O)OR⁷, C₁₋₃alkyleneheteroaryl, C(═O)C(═O)OR⁷,C(═O)Cl₃alkyleneC(═O)OR⁷, C(═O)C₁₋₃alkyleneNHC(═O)OR⁷, C(═O)C₁₋₃alkyleneNH₂, and NHC(═O)OR⁷;

[0033] R⁴ is hydrogen, lower alkyl, haloalkyl, cycloalkyl, aryl,C(═O)R⁷, OR⁸, NR⁸R⁹, or SR⁸;

[0034] R⁵ is hydrogen, lower alkyl, haloalkyl, cycloalkyl, or aryl;

[0035] R⁷ is selected from the group consisting of cycloalkyl, branchedor unbranched lower alkyl, heteroaryl, a heterocycle, aralkyl, and aryl,and R⁷ is optionally substituted with one or more of OR⁸, NR⁸R⁹, or SR⁸;and

[0036] R⁸ and R⁹, same or different, are selected from the groupconsisting of hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl,alkaryl, heteroaralkyl, heteroalkaryl, and aralkyl, or R⁸ and R⁹ aretaken together form a 4-membered to 7-membered ring;

[0037] R¹⁰ is hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, C(═O)alkyl,C(═O)cycloalkyl, C(═O)aryl, C(═O)Oalkyl, C(═O)Ocycloalkyl, C(═O)aryl,CH₂OH, CH₂Oalkyl, CHO, CN, NO₂, or SO₂R¹¹; and

[0038] R¹¹ is alkyl, cycloalkyl, trifluoromethyl, aryl, aralkyl, orNR⁸R⁹.

[0039] A compound of structural formula (II) can be used alone, eitherneat or in a composition that further contains a pharmaceuticallyacceptable carrier. A compound of structural formula (II) also can beadministering in conjunction with a second active therapeutic agent, forexample, a second antiinflammatory therapeutic agent, like an agentcapable of targeting TNFα.

[0040] Compounds of structural formula (II) can be used to modulate cAMPlevels in a mammal, reduce TNFα levels in a mammal, suppressinflammatory cell activation in a mammal, inhibit PDE4 function in amammal, and treat conditions afflicting a mammal where inhibition ofPDE4 provides a benefit.

[0041] As used herein, the term “alkyl,” alone or in combination, isdefined to include straight chain and branched chain, and bridged,saturated hydrocarbon groups containing one to 16 carbon atoms. The term“lower alkyl” is defined herein as an alkyl group having one through sixcarbon atoms (C₁-C₆). Examples of lower alkyl groups include, but arenot limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl,neopentyl, n-hexyl, and the like. The term “alkynyl” refers to anunsaturated alkyl group that contains a carbon-carbon triple bond.

[0042] The term “bridged alkyl” is defined herein as a C₆-C₁₆ bicyclicor polycyclic hydrocarbon group, for example, norboryl, adamantyl,bicyclo [2.2.2]-octyl, bicyclo [2.2.] heptyl, bicyclo [3.2.1] octyl, ordecahydronaphthyl.

[0043] The term “allyl” is defined herein as CH₂═CH—CH₂—, optionallysubstituted, e.g., alkyl or aryl substituted.

[0044] The term “propargyl” is defined herein as CH≡C—CH₂—, optionallysubstituted, e.g., alkyl or aryl substituted.

[0045] The term “cycloalkyl” is defined herein to include cyclic C₃-C₇hydrocarbon groups. Examples of cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.

[0046] The term “alkylene” refers to an alkyl group having asubstituent. For example, the term “C₁₋₃alkylenecycloalkyl” refers to analkyl group containing one to three carbon atoms, and substituted with acycloalkyl group.

[0047] The term “haloalkyl” is defined herein as an alkyl groupsubstituted with one or more halo substituents, either fluro, chloro,bromo, iodo, or combinations thereof. Similarly, “halocycloalkyl” isdefined as a cycloalkyl group having one or more halo substituents.

[0048] The term “aryl,” alone or in combination, is defined herein as amonocyclic or polycyclic aromatic group, preferably a monocyclic orbicyclic aromatic group, e.g., phenyl or naphthyl, that can beunsubstituted or substituted, for example, with one or more, and inparticular one to three, substituents selected from halo, alkyl,hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, alkoxyalkyl,aryloxyalkyl, aralkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Exemplary arylgroups include phenyl, naphthyl, tetrahydronaphthyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl,3-trifluoromethyl-phenyl, 4-nitrophenyl, and the like.

[0049] The term “heteroaryl” is defined herein as a monocyclic orbicyclic ring system containing one or two aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring, andwhich can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, substituents, like halo, alkyl,hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, alkoxy-alkyl,aryloxyalkyl, aralkoxyalkyl, haloalkyl, nitro, amino, alkylamino,acylamino, alkylthio, alkyl-sulfinyl, and alkylsulfonyl. Examples ofheteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl,isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0050] The term “aralkyl” is defined herein as a previously definedalkyl group, wherein one of the hydrogen atoms is replaced by an arylgroup as defined herein, for example, a phenyl group optionally havingone or more substituents, for example, halo, alkyl, alkoxy, and thelike. An example of an aralkyl group is a benzyl group.

[0051] The term “alkaryl” is defined herein as a previously defined arylgroup, wherein one of the hydrogen atoms is replaced by an alkyl,cycloalkyl, haloalkyl, or halocycloalkyl group.

[0052] The terms “heteroaralkyl” and “heteroalkaryl” are definedsimilarly as the term “aralkyl” and “alkaryl,” however, the aryl groupis replaced by a heteroaryl group as previously defined.

[0053] The term “heterocycle” is defined as a 5-or 6-memberednonaromatic ring, optionally substituted, having one or more heteroatomsselected from oxygen, nitrogen, and sulfur present in the ring.Nonlimiting examples include tetrahydrofuran, piperidine, piperazine,sulfolane, morpholine, tetrahydropyran, dioxane, and the like.

[0054] The term “halogen” or “halo” is defined herein to includefluorine, chlorine, bromine, and iodine.

[0055] The terms “alkoxy,” “aryloxy,” and “aralkoxy” are defined as —OR,wherein R is alkyl, aryl, and aralkyl, respectively.

[0056] The term “alkoxyalkyl” is defined as an alkoxy group appended toan alkyl group. The terms “aryloxyalkyl” and “aralkoxyalkyl” aresimilarly defined as an aryloxy or aralkoxy group appended to an alkylgroup.

[0057] The term “hydroxy” is defined as —OH.

[0058] The term “hydroxyalkyl” is defined as a hydroxy group appended toan alkyl group.

[0059] The term “amino” is defined as —NH₂.

[0060] The term “alkylamino” is defined as —NR₂ wherein at least one Ris alkyl and the second R is alkyl or hydrogen.

[0061] The term “acylamino” is defined as RC(═O)N, wherein R is alkyl oraryl.

[0062] The term “nitro” is defined as —NO₂.

[0063] The term “alkylthio” is defined as —SR, where R is alkyl.

[0064] The term “alkylsulfinyl” is defined as R—SO₂, where R is alkyl.

[0065] The term “alkylsulfonyl” is defined as R—SO₃, where R is alkyl.

[0066] In preferred embodiments, R⁵ is hydrogen, R⁷ is methyl, R² ismethyl or difluoromethyl, R⁴ is selected from the group consisting ofhydrogen, methyl, trifluoromethyl, cyclopropyl, acetyl, ethynyl,benzoyl, phenyl, and NR⁸R⁹, wherein R⁸ and R⁹, independently, arehydrogen, lower alkyl, or cycloalkyl, or are taken together to form a5-membered or 6-membered ring. R¹ is selected from the group consistingof

[0067] R³ is selected from the group consisting of

[0068] In most preferred embodiments, R¹ is selected from the groupconsisting of cyclopentyl, tetrahydrofuryl, indanyl, norbornyl,phenethyl, and phenylbutyl; R² is selected from the group consisting ofmethyl and difluoromethyl; R³ is selected from the group consisting ofCO₂CH₃, C(═O)CH₂OH, C(═O)CH(CH₃)—OH, C(═O)C(CH₃)₂OH, and

[0069] R⁴ is NR⁸R⁹; R⁵ is hydrogen; R⁷ is methyl; R⁸ and R⁹,independently, are selected from the group consisting of hydrogen andlower alkyl, or are taken together form a 5-membered or 6-membered ring;and R¹⁰ is hydrogen.

[0070] The present invention includes all possible stereoisomers andgeometric isomers of compounds of structural formula (II), and includesnot only racemic compounds but also the optically active isomers aswell. When a compound of structural formula (II) is desired as a singleenantiomer, it can be obtained either by resolution of the final productor by stereospecific synthesis from either isomerically pure startingmaterial or use of a chiral auxiliary reagent, for example, see Z. Ma etal., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution ofthe final product, an intermediate, or a starting material can beachieved by any suitable method known in the art. Additionally, insituations where tautomers of the compounds of structural formula (II)are possible, the present invention is intended to include alltautomeric forms of the compounds. As demonstrated hereafter, specificstereoisomers exhibit an exceptional ability to inhibit PDE4 withoutmanifesting the adverse CNS side effects typically associated with PDE4inhibitors.

[0071] In particular, it is generally accepted that biological systemscan exhibit very sensitive activities with respect to the absolutestereochemical nature of compounds. (See, E. J. Ariens, MedicinalResearch Reviews, 6:451-466 (1986); E. J. Ariens, Medicinal ResearchReviews, 7:367-387 (1987); K. W. Fowler, Handbook of Stereoisomers:Therapeutic Drugs, CRC Press, edited by Donald P. Smith, pp. 35-63(1989); and S. C. Stinson, Chemical and Engineering News, 75:38-70(1997).)

[0072] For example, rolipram is a stereospecific PDE4 inhibitor thatcontains one chiral center. The (−)-enantiomer of rolipram has a higherpharmacological potency than the (+)-enantiomer, which could be relatedto its potential antidepressant action. Schultz et al.,Naunyn-Schmiedeberg's Arch Pharmacol, 333:23-30 (1986). Furthermore, themetabolism of rolipram appears stereospecific with the (+)-enantiomerexhibiting a faster clearance rate than the (−)-enantiomer. Krause etal., Xenobiotica, 18:561-571 (1988). Finally, a recent observationindicated that the (−)-enantiomer of rolipram (R-rolipram) is aboutten-fold more emetic than the (+)-enantiomer (S-rolipram). A. Robichaudet al., Neuropharmacology, 38:289-297 (1999). This observation is noteasily reconciled with differences in test animal disposition torolipram isomers and the ability of rolipram to inhibit the PDE4 enzyme.The compounds of the present invention can have three chiral centers. Asshown below, compounds of a specific stereochemical orientation exhibitsimilar PDE4 inhibitory activity and pharmacological activity, butaltered CNS toxicity and emetic potential.

[0073] Accordingly, preferred compounds of the present invention havethe structural formula (III):

[0074] The compounds of structural formula (III) are potent andselective PDE4 inhibitors, and do not manifest the adverse CNS effectsand emetic potential demonstrated by stereoisomers of a compound ofstructural formula (III).

[0075] Compounds of structural formula (II) which contain acidicmoieties can form pharmaceutically acceptable salts with suitablecations. Suitable pharmaceutically acceptable cations include alkalimetal (e.g., sodium or potassium) and alkaline earth metal (e.g.,calcium or magnesium) cations. The pharmaceutically acceptable salts ofthe compounds of structural formula (II), which contain a basic center,are acid addition salts formed with pharmaceutically acceptable acids.Examples include the hydrochloride, hydrobromide, sulfate or bisulfate,phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate,maleate, lactate, citrate, tartrate, gluconate, methanesulfonate,benzenesulphonate, and p-toluenesulphonate salts. In light of theforegoing, any reference to compounds of the present invention appearingherein is intended to include compounds of structural formula (II), aswell as pharmaceutically acceptable salts and solvates thereof.

[0076] The compounds of the present invention can be therapeuticallyadministered as the neat chemical, but it is preferable to administercompounds of structural formula (II) as a pharmaceutical composition orformulation. Accordingly, the present invention further provides forpharmaceutical formulations comprising a compound of structural formula(II), together with one or more pharmaceutically acceptable carriersand, optionally, other therapeutic and/or prophylactic ingredients. Thecarriers are “acceptable” in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

[0077] In particular, a selective PDE4 inhibitor of the presentinvention is useful alone or in combination with a secondantiinflammatory therapeutic agent, for example, a therapeutic agenttargeting TNFα, such as ENBREL® or REMICADE®, which have utility intreating rheumatoid arthritis. Likewise, therapeutic utility of IL-1antagonism has also been shown in animal models for rheumatoidarthritis. Thus, it is envisioned that IL-1 antagonism, in combinationwith PDE4 inhibition, which attenuates TNFα, would be efficacious.

[0078] The present PDE4 inhibitors are useful in the treatment of avariety of allergic, autoimmune, and inflammatory diseases.

[0079] The term “treatment” includes preventing, lowering, stopping, orreversing the progression of severity of the condition or symptoms beingtreated. As such, the term “treatment” includes both medical therapeuticand/or prophylactic administration, as appropriate.

[0080] In particular, inflammation is a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute or wall off (i.e., sequester) both the injurious agent and theinjured tissue. The term “inflammatory disease,” as used herein, meansany disease in which an excessive or unregulated inflammatory responseleads to excessive inflammatory symptoms, host tissue damage, or loss oftissue function. Additionally, the term “autoimmune disease,” as usedherein, means any group of disorders in which tissue injury isassociated with humoral or cell-mediated responses to the body's ownconstituents. The term “allergic disease,” as used herein, means anysymptoms, tissue damage, or loss of tissue function resulting fromallergy. The term “arthritic disease,” as used herein, means any of alarge family of diseases that are characterized by inflammatory lesionsof the joints attributable to a variety of etiologies. The term“dermatitis,” as used herein, means any of a large family of diseases ofthe skin that are characterized by inflammation of the skin attributableto a variety of etiologies. The term “transplant rejection,” as usedherein, means any immune reaction directed against grafted tissue(including organ and cell (e.g., bone marrow)), characterized by a lossof function of the grafted and surrounding tissues, pain, swelling,leukocytosis and thrombocytopenia.

[0081] The present invention also provides a method of modulating cAMPlevels in a mammal, as well as a method of treating diseasescharacterized by elevated cytokine levels.

[0082] The term “cytokine,” as used herein, means any secretedpolypeptide that affects the functions of other cells, and thatmodulates interactions between cells in the immune or inflammatoryresponse. Cytokines include, but are not limited to monokines,lymphokines, and chemokines regardless of which cells produce them. Forinstance, a monokine is generally referred to as being produced andsecreted by a monocyte, however, many other cells produce monokines,such as natural killer cells, fibroblasts, basophils, neutrophils,endothelial cells, brain astrocytes, bone marrow stromal cells,epidermal keratinocytes, and B-lymphocytes. Lymphokines are generallyreferred to as being produced by lymphocyte cells. Examples of cytokinesinclude, but are not limited to, interleukin-1 (IL-1), interleukin-6(IL-6), Tumor Necrosis Factor alpha (TNFα), and Tumor Necrosis Factorbeta (TNFβ).

[0083] The present invention further provides a method of reducing TNFlevels in a mammal, which comprises administering an effective amount ofa compound of structural formula (II) to the mammal. The term “reducingTNF levels,” as used herein, means either:

[0084] a) decreasing excessive in vivo TNF levels in a mammal to normallevels or below normal levels by inhibition of the in vivo release ofTNF by all cells, including but not limited to monocytes or macrophages;or

[0085] b) inducing a down-regulation, at the translational ortranscription level, of excessive in vivo TNF levels in a mammal tonormal levels or below normal levels; or

[0086] c) inducing a down-regulation, by inhibition of the directsynthesis of TNF as a postranslational event.

[0087] Moreover, the compounds of the present invention are useful insuppressing inflammatory cell activation. The term “inflammatory cellactivation,” as used herein, means the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes, polymorphonuclearleukocytes, mast cells, basophils, eosinophils, dendritic cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

[0088] The compounds of the present invention also are useful in causingairway smooth muscle relaxation, bronchodilation, and prevention ofbronchoconstriction.

[0089] The compounds of the present invention, therefore, are useful intreating such diseases as arthritic diseases (such as rheumatoidarthritis), osteoarthritis, gouty arthritis, spondylitis,thyroid-associated ophthalmopathy, Behcet disease, sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shocksyndrome, asthma, chronic bronchitis, allergic rhinitis, allergicconjunctivitis, vernal conjunctivitis, eosinophilic granuloma, adult(acute) respiratory distress syndrome (ARDS), chronic pulmonaryinflammatory disease (such as chronic obstructive pulmonary disease),silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium,brain or extremities, brain or spinal cord injury due to minor trauma,fibrosis including cystic fibrosis, keloid formation, scar tissueformation, atherosclerosis, autoimmune diseases, such as systemic lupuserythematosus (SLE) and transplant rejection disorders (e.g., graft vs.host (GvH) reaction and allograft rejection), chronicglomerulonephritis, inflammatory bowel diseases, such as Crohn's diseaseand ulcerative colitis, proliferative lymphocytic diseases, such asleukemias (e.g. chronic lymphocytic leukemia; CLL) (see Mentz et al.,Blood 88, pp. 2172-2182 (1996)), and inflammatory dermatoses, such asatopic dermatitis, psoriasis, or urticaria.

[0090] Other examples of such diseases or related conditions includecardiomyopathies, such as congestive heart failure, pyrexia, cachexia,cachexia secondary to infection or malignancy, cachexia secondary toacquired immune deficiency syndrome (AIDS), ARC (AIDS-related complex),cerebral malaria, osteoporosis and bone resorption diseases, and feverand myalgias due to infection. In addition, the compounds of the presentinvention are useful in the treatment of diabetes insipidus and centralnervous system disorders, such as depression and multi-infarct dementia.

[0091] Compounds of the present invention also have utility outside ofthat typically known as therapeutic. For example, the present compoundscan function as organ transplant preservatives (see Pinsky et al., J.Clin. Invest., 92, pp. 2994-3002 (1993)) as well.

[0092] Selective PDE4 inhibitors also can be useful in the treatment oferectile dysfunction, especially vasculogenic impotence (Doherty, Jr. etal. U.S. Pat. No. 6,127,363), diabetes insipidus (Kidney Int., 37, p.362, (1990); Kidney Int., 35, p. 494, (1989)) and central nervous systemdisorders, such as multiinfarct dementia (Nicholson, Psychopharmacology,101, p. 147 (1990)), depression (Eckman et al., Curr. Ther. Res., 43, p.291 (1988)), anxiety and stress responses (Neuropharmacology, 38, p.1831 (1991)), cerebral ischemia (Eur. J. Pharmacol., 272, p. 107(1995)), tardive dyskinesia (J. Clin. Pharmocol., 16, p. 304 (1976)),Parkinson's disease (see Neurology, 25, p. 722 (1975); Clin. Exp.Pharmacol, Physiol., 26, p. 421 (1999)), and premenstrual syndrome. Withrespect to depression, PDE4-selective inhibitors show efficacy in avariety of animal models of depression such as the “behavioral despair”or Porsolt tests (Eur. J. Pharmacol., 47, p. 379 (1978); Eur. J.Pharmacol., 57, p. 431 (1979); Antidepressants: neurochemical,behavioral and clinical prospectives, Enna, Malick, and Richelson, eds.,Raven Press, p. 121 (1981)), and the “tail suspension test”(Psychopharmacology, 85, p. 367 (1985)). Recent research findings showthat chronic in vivo treatment by a variety of antidepressants increasethe brain-derived expression of PDE4 (J. Neuroscience, 19, p. 610(1999)). Therefore, a selective PDE4 inhibitor can be used alone or inconjunction with a second therapeutic agent in a treatment for the fourmajor classes of antidepressants: electroconvulsive procedures,monoamine oxidase inhibitors, and selective reuptake inhibitors ofserotonin or norepinephrine. Selective PDE4 inhibitors also can beuseful in applications that modulate bronchodilatory activity via directaction on bronchial smooth muscle cells for the treatment of asthma.

[0093] Compounds and pharmaceutical compositions suitable for use in thepresent invention include those wherein the active ingredient isadministered to a mammal in an effective amount to achieve its intendedpurpose. More specifically, a “therapeutically effective amount” meansan amount effective to prevent development of, or to alleviate theexisting symptoms of, the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

[0094] The term “mammal” as used herein includes males and females, andencompasses humans, domestic animals (e.g., cats, dogs), livestock(e.g., cattle, horses, swine), and wildlife (e.g., primates, large cats,zoo specimens).

[0095] A “therapeutically effective dose” refers to that amount of thecompound that results in achieving the desired effect. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀. (the dose lethal to 50% of thepopulation) and the ED₅₀. (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, which is expressed as the ratio between LD₅₀andED₅₀. Compounds which exhibit high therapeutic indices are preferred.The data obtained from such data can be used in formulating a dosagerange for use in humans. The dosage of such compounds preferably lieswithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed, and the route of administration utilized.

[0096] The exact formulation, route of administration, and dosage can bechosen by the individual physician in view of the patient's condition.Dosage amount and interval can be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thetherapeutic effects.

[0097] As appreciated by persons skilled in the art, reference herein totreatment extends to prophylaxis, as well as to treatment of establisheddiseases or symptoms. It is further appreciated that the amount of acompound of the invention required for use in treatment varies with thenature of the condition being treated, and with the age and thecondition of the patient, and is ultimately determined by the attendantphysician or veterinarian. In general, however, doses employed for adulthuman treatment typically are in the range of 0.001 mg/kg to about 100mg/kg per day. The desired dose can be conveniently administered in asingle dose, or as multiple doses administered at appropriate intervals,for example as two, three, four or more subdoses per day. In practice,the physician determines the actual dosing regimen which is mostsuitable for an individual patient, and the dosage varies with the age,weight, and response of the particular patient. The above dosages areexemplary of the average case, but there can be individual instances inwhich higher or lower dosages are merited, and such are within the scopeof the present invention.

[0098] Formulations of the present invention can be administered in astandard manner for the treatment of the indicated diseases, such asorally, parenterally, transmucosally (e.g., sublingually or via buccaladministration), topically, transdermally, rectally, via inhalation(e.g., nasal or deep lung inhalation). Parenteral administrationincludes, but is not limited to intravenous, intra-arterial,intraperitoneal, subcutaneous, intramuscular, intrathecal, andintraarticular. Parenteral administration also can be accomplished usinga high pressure technique, like POWDERJECT™.

[0099] For buccal administration, the composition can be in the form oftablets or lozenges formulated in conventional manner. For example,tablets and capsules for oral administration can contain conventionalexcipients such as binding agents (for example, syrup, accacia, gelatin,sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone),fillers (for example, lactose, sugar, microcrystalline, cellulose,maize-starch, calcium phosphate or sorbitol), lubricants (for example,magnesium, stearate, stearic acid, talc, polyethylene glycol or silica),disintegrants (for example, potato starch or sodium starch glycollate),or wetting agents (for example, sodium lauryl sulfate). The tablets canbe coated according to methods well known in the art.

[0100] Alternatively, the compounds of the present invention can beincorporated into oral liquid preparations such as aqueous or oilysuspensions, solutions, emulsions, syrups, or elixirs, for example.Moreover, formulations containing these compounds can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can contain conventional additives, suchas suspending agents, such as sorbitol syrup, methyl cellulose,glucose/sugar syrup, gelatin, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethyl cellulose, aluminum stearategel, and hydrogenated edible fats; emulsifying agents, such as lecithin,sorbitan mono-oleate, or acacia; nonaqueous vehicles (which can includeedible oils), such as almond oil, fractionated coconut oil, oily esters,propylene glycol, and ethyl alcohol; and preservatives, such as methylor propyl p-hydroxybenzoate and sorbic acid.

[0101] Such preparations also can be formulated as suppositories, e.g.,containing conventional suppository bases, such as cocoa butter or otherglycerides. Compositions for inhalation typically can be provided in theform of a solution, suspension, or emulsion that can be administered asa dry powder or in the form of an aerosol using a conventionalpropellant, such as dichlorodifluoromethane or trichlorofluoromethane.Typical topical and transdermal formulations comprise conventionalaqueous or nonaqueous vehicles, such as eye drops, creams, ointments,lotions, and pastes, or are in the form of a medicated plaster, patch,or membrane.

[0102] Additionally, compositions of the present invention can beformulated for parenteral administration by injection or continuousinfusion. Formulations for injection can be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulation agents, such as suspending, stabilizing, and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water)before use.

[0103] A composition in accordance with the present invention also canbe formulated as a depot preparation. Such long acting formulations canbe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Accordingly, thecompounds of the invention can be formulated with suitable polymeric orhydrophobic materials (e.g., an emulsion in an acceptable oil), ionexchange resins, or as sparingly soluble derivatives (e.g., a sparinglysoluble salt).

[0104] For veterinary use, a compound of formula (II), or nontoxic saltsthereof, is administered as a suitably acceptable formulation inaccordance with normal veterinary practice. The veterinarian can readilydetermine the dosing regimen and route of administration that is mostappropriate for a particular animal.

[0105] Thus, the invention provides in a further aspect a pharmaceuticalcomposition comprising a compound of the formula (II), together with apharmaceutically acceptable diluent or carrier therefor. There isfurther provided by the present invention a process of preparing apharmaceutical composition comprising a compound of formula (II), whichprocess comprises mixing a compound of formula (II), together with apharmaceutically acceptable diluent or carrier therefor.

[0106] A specific, nonlimiting example of a compound of structuralformula (II) is provided below, the synthesis of which was performed inaccordance with the procedure set forth below.

[0107] Generally, compounds of structural formula (II) can be preparedaccording to the following synthetic scheme. In the scheme describedbelow, it is understood in the art that protecting groups can beemployed where necessary in accordance with general principles ofsynthetic chemistry. These protecting groups are removed in the finalsteps of the synthesis under basic, acidic, or hydrogenolytic conditionswhich are readily apparent to those skilled in the art. By employingappropriate manipulation and protection of any chemical functionalities,synthesis of compounds of structural formula (II) not specifically setforth herein can be accomplished by methods analogous to the schemes setforth below.

[0108] Unless otherwise noted, all starting materials were obtained fromcommercial suppliers and used without further purification. Allreactions and chromatography fractions were analyzed by thin-layerchromatography on 250-mm silica gel plates, visualized with UV(ultraviolet) light or I₂ (iodine) stain. Products and intermediateswere purified by flash chromatography, or reverse-phase HPLC.

[0109] The compounds of general structural formula (II) can be prepared,for example, as set forth in the following Example 1. Other syntheticroutes also are known to persons skilled in the art. The followingreaction scheme provides a compound of structural formula (II), whereinR¹ and R², i.e., cyclopentyl and CH₃, are determined by the startingmaterials. Proper selection of other starting materials, or performingconversion reactions on intermediates and examples, provide compounds ofgeneral structural formula (II) having other recited R¹ through R¹¹substituents. The following example is provided for illustration andshould not be construed as limiting.

EXAMPLE 1

[0110] Preparation of (3S,4R)-4-(3-Cyclopentyloxy-4-methoxyphenyl)-3-methyl-3-(2-methylaminothiazol-4-yl)pyrrolidine-1-carboxylicacid methyl ester

[0111] To a solution of 1-methyl-2-thiourea (0.038 g, 0.418 mmol) inmethanol (1 mL), at 50° C., was added a solution of (3S,4R)-3-(2-bromoethanoyl)-4-(3-cyclopentyloxy-4-methoxyphenyl)-3-methyl-pyrrolidine-1-carboxylicacid methyl ester (0.19 g, 0.42 mmol) in methanol (1 mL). Thetemperature was raised to 70° C. and refluxed for 2 hours. The reactionmixture then was concentrated under reduced pressure and dissolved inacetonitrile-water (CH₃CN-H₂O ) (1 mL) for purification by HPLC on a C18column (Luna 10 μ, C18, 250×10 mm). Gradient elution of 50-100%acetonitrile-water (0.05% TFA) yielded the named product as a whitepowder after lyophilization (91 mg, 49%).

[0112]¹H NMR (300 MHz, CDCl₃) δ (ppm): 11.84 (s, 1H, NH) 6.95-6.70 (m,3H, aromatic), 6.14 (m, 1H, aromatic), 4.87-4.82 (br. m, 1H), 3.97-3.5(m, 4H), 3.84 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.40-3.36 (m, 1H), 3.05(s, 3H, NCH₃), 2.0-1.6 (m, 8H, cyclopentyl), 1.07 (s, 3H, CH₃).

[0113] The compounds of structural formula (II) were tested for anability to inhibit PDE4. The ability of a compound to inhibit PDE4activity is related to the IC₅₀ value for the compound, i.e., theconcentration of inhibitor required for 50% inhibition of enzymeactivity. The IC₅₀ value for compounds of structural formula (II) weredetermined using recombinant human PDE4.

[0114] The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE4 of less than about 100 μM, andpreferably less than about 50 μM, and more preferably less than about 25μm. The compounds of the present invention typically exhibit an IC₅₀value against recombinant human PDE4 of less than about 5 μM, and oftenless than about 1 μM. To achieve the full advantage of the presentinvention, a present PDE4 inhibitor has an IC₅₀ of about 0.05 μM toabout 15 μM.

[0115] The IC₅₀ values for the compounds were determined fromconcentration-response curves typically using concentrations rangingfrom 0.1 μM to 500 μM. Tests against other PDE enzymes using standardmethodology, as described in Loughney et al., J. Biol. Chem., 271, pp.796-806 (1996), also showed that compounds of the present invention arehighly selective for the cAMP-specific PDE4 enzyme.

[0116] The production of recombinant human PDEs and the IC₅₀determinations can be accomplished by well-known methods in the art.Exemplary methods are described as follows:

[0117] EXPRESSION OF HUMAN PDEs

[0118] Expression in Baculovirus-Infected Spodoptera fugiperda (Sf9)Cells

[0119] Baculovirus transfer plasmids were constructed using eitherpBlueBacIII (Invitrogen) or pFastBac (BRL-Gibco). The structure of allplasmids was verified by sequencing across the vector junctions and byfully sequencing all regions generated by PCR. Plasmid pBB-PDE1A3/6contained the complete open reading frame of PDE1A3 (Loughney et al., J.Biol. Chem., 271, pp. 796-806 (1996)) in pBlue-BacIII. Plasmid Hcam3aBBcontained the complete open reading frame of PDE1C3 (Loughney et al.(1996)) in pBlueBacIII. Plasmid pBB-PDE3A contained the complete openreading frame of PDE3A (Meacci et al., Proc. Natl. Acad. Sci., USA, 89,pp. 3721-3725 (1992)) in pBlueBacIII.

[0120] Recombinant virus stocks were produced using either the MaxBacsystem (Invitrogen) or the FastBac system (Gibco-BRL) according to themanufacturer's protocols. In both cases, expression of recombinant humanPDEs in the resultant viruses was driven off the viral polyhedronpromoter. When using the MaxBac® system, virus was plaque purified twicein order to insure that no wild type (occ+) virus contaminated thepreparation. Protein expression was carried out as follows. Sf9 cellswere grown at 27° C. in Grace's Insect culture medium (Gibco-BRL)supplemented with 10% fetal bovine serum, 0.33% TC yeastolate, 0.33%lactalbumin hydrolysate, 4.2 mM NaHCO₃, 10 μg/mL gentamycin, 100units/mL penicillin, and 100 μg/mL streptomycin. Exponentially growingcells were infected at a multiplicity of approximately 2 to 3 virusparticles per cell and incubated for 48 hours. Cells were collected bycentrifugation, washed with nonsupplemented Grace's medium, andquick-frozen for storage.

[0121] Expression in Saccharomyces cerevisiae (Yeast)

[0122] Recombinant production of human PDE1B, PDE2, PDE4A, PDE4B, PDE4C,PDE4D, PDE5, and PDE7 was carried out similarly to that described inExample 7 of U.S. Pat. No. 5,702,936, incorporated herein by reference,except that the yeast transformation vector employed, which is derivedfrom the basic ADH2 plasmid described in Price et al., Methods inEnzymology, 185, pp. 308-318 (1990), incorporated yeast ADH2 promoterand terminator sequences and the Saccharomyces cerevisiae host was theprotease-deficient strain BJ2-54 deposited on Aug. 31, 1998 with theAmerican Type Culture Collection, Manassas, Va., under accession numberATCC₇₄₄₆₅. Transformed host cells were grown in 2× SC-leu medium, pH6.2, with trace metals, and vitamins. After 24 hours, YEPmedium-containing glycerol was added to a final concentration of 2×YET/3% glycerol. Approximately 24 hr later, cells were harvested,washed, and stored at −70° C.

[0123] HUMAN PHOSPHODIESTERASE PREPARATIONS

[0124] Phosphodiesterase Activity Determinations

[0125] Phosphodiesterase activity of the preparations was determined asfollows. PDE assays utilizing a charcoal separation technique wereperformed essentially as described in Loughney et al. (1996). In thisassay, PDE activity converts [32P]cAMP or [32P]cGMP to the corresponding[32P]5′-AMP or [32P]5′-GMP in proportion to the amount of PDE activitypresent. The [32P]5′-AMP or [32P]5′-GMP then was quantitativelyconverted to free [32P]phosphate and unlabeled adenosine or guanosine bythe action of snake venom 5′-nucleotidase. Hence, the amount of[32P]phosphate liberated is proportional to enzyme activity. The assaywas performed at 30° C. in a 100 μL reaction mixture containing (finalconcentrations) 40 mM Tris HCl (pH 8.0), 1 μM ZnSO₄, 5 mM MgCl₂, and 0.1mg/mL bovine serum albumin (BSA). Alternatively, in assays assessingPDE1-specific activity, incubation mixtures further incorporated the useof 0.1 mM CaCl₂ and 10 μg/mL calmodulin. PDE enzyme was present inquantities that yield<30% total hydrolysis of substrate (linear assayconditions). The assay was initiated by addition of substrate (1 mM[32P]cAMP or cGMP), and the mixture was incubated for 12 minutes.Seventy-five (75) μg of Crotalus atrox venom then was added, and theincubation was continued for 3 minutes (15 minutes total). The reactionwas stopped by addition of 200 μL of activated charcoal (25 mg/mLsuspension in 0.1 M NaH₂pH4, pH 4). After centrifugation (750× g for 3minutes) to sediment the charcoal, a sample of the supernatant was takenfor radioactivity determination in a scintillation counter and the PDEactivity was calculated.

[0126] Inhibitor analyses were performed similarly to the methoddescribed in Loughney et al., J. Biol. Chem., 271, pp. 796-806 (1996),except both cGMP and cAMP were used, and substrate concentrations werekept below 32 nM, which is far below the Km of the tested PDEs.

[0127] Human PDE4A, 4B, 4C, 4D Preparations

[0128] Preparation of PDE4A from S. cerevisiae

[0129] Yeast cells (50 g of yeast strain YI26 harboring HDUN1.46) werethawed at room temperature by mixing with 50 mL of Lysis Buffer (50 mMMOPS pH 7.5, 10 μM ZnSO₄, 2 mM MgCl₂₁ 14.2 mM 2-mercaptoethanol, 5 μg/mLeach of pepstatin, leupeptin, aprotinin, 20 μg/mL each of calpaininhibitors I and II, and 2 mM benzamidine HCl). Cells were lysed in aFrench® pressure cell (SLM-Aminco®, Spectronic Instruments) at 10° C.The extract was centrifuged in a Beckman JA-10 rotor at 9,000 rpm for 22minutes at 4° C. The supernatant was removed and centrifuged in aBeckman TI45 rotor at 36,000 rpm for 45 minutes at 4° C.

[0130] PDE4A was precipitated from the high-speed supernatant by theaddition of solid ammonium sulfate (0.26 g/mL supernatant) whilestirring in an ice bath and maintaining the pH between 7.0 and 7.5. Theprecipitated proteins containing PDE4A were collected via centrifugationin a Beckman JA-10 rotor at 9,000 rpm for 22 minutes. The precipitatewas resuspended in 50 mL of Buffer G (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5MM MgCl₂, 100 mM NaCl, 14.2 mM 2-mercaptoethanol, 2 mM benzamidine HCl,5 μg/mL each of leupeptin, pepstatin, and aprotinin, and 20 μg/mL eachof calpain inhibitors I and II) and passed through a 0.45 μm filter.

[0131] The resuspended sample (50 to 100 mL) was loaded onto a 5×100 cmcolumn of Pharmacia SEPHACRYL® S-300 equilibrated in Buffer G. Enzymeactivity was eluted at a flow rate of 2 mL/min and pooled for laterfractionation.

[0132] The PDE4A isolated from gel filtration chromatography was appliedto a 1.6×20 cm column of Sigma Cibacron Blue Agarose-type 300 (10 mL)equilibrated in Buffer A (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5 mM MgCl₂,14.2 mM 2-mercaptoethanol, and 100 mM benzamidine HCl). The column waswashed in succession with 50 to 100 mL of Buffer A, 20 to 30 mL ofBuffer A containing 20 mM 5′-AMP, 50 to 100 mL of Buffer A containing1.5 M NaCl, and 10 to 20 mL of Buffer C (50 mM Tris HCl pH 8, 10 μMZnSO₄, 14.2 mM 2-mercaptoethanol, and 2 mM benzamidine HCl). The enzymewas eluted with 20 to 30 mL of Buffer C containing 20 mM cAMP.

[0133] The PDE activity peak was pooled, and precipitated with ammoniumsulfate (0.33 g/mL enzyme pool) to remove excess cyclic nucleotide. Theprecipitated proteins were resuspended in Buffer X (25 mM MOPS pH 7.5, 5μM ZnSO₄, 50 mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl), and desaltedvia gel filtration on a Pharmacia PD-10® column per manufacturer'sinstructions. The enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

[0134] The resultant preparations were about >80% pure by SDS-PAGE.These preparations had specific activities of about 10 to 40 μmol cAMPhydrolyzed per minute per milligram protein.

[0135] Preparation of PDE4B from S. cerevisiae

[0136] Yeast cells (150 g of yeast strain YI23 harboring HDUN2.32) werethawed by mixing with 100 mL glass beads (0.5 mM, acid washed) and 150mL Lysis Buffer (50 mM MOPS pH 7.2, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mMbenzamidine HCl, 5 μg/mL each of pepstatin, leupeptin, aprotinin,calpain inhibitors I and II) at room temperature. The mixture was cooledto 4° C., transferred to a Bead-Beater®, and the cells lysed by rapidmixing for 6 cycles of 30 seconds each. The homogenate was centrifugedfor 22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at9,000 rpm and 4° C. The supernatant was recovered and centrifuged in aBeckman XL-80 ultra-centrifuge using a TI45 rotor at 36,000 rpm for 45minutes at 4° C. The supernatant was recovered and PDE4B wasprecipitated by the addition of solid ammonium sulfate (0.26 g/mLsupernatant) while stirring in an ice bath and maintaining the pHbetween 7.0 and 7.5. This mixture was then centrifuged for 22 minutes ina Beckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000× g).The supernatant was discarded and the pellet was dissolved in 200 mL ofBuffer A (50 mM MOPS pH 7.5, 5 mM MgCl₂₁ 1 mM DTT, 1 mM benzamidine HCl,and 5 μg/mL each of leupeptin, pepstatin, and aprotinin). The pH andconductivity were corrected to 7.5 and 15-20 mS, respectively.

[0137] The resuspended sample was loaded onto a 1.6×200 cm column (25mL) of Sigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A.The sample was cycled through the column 4 to 6 times over the course of12 hours. The column was washed in succession with 125 to 250 mL ofBuffer A, 125 to 250 mL of Buffer A containing 1.5 M NaCl, and 25 to 50mL of Buffer A. The enzyme was eluted with 50 to 75 mL of Buffer E (50mM Tris HCl pH 8, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl,and 20 mM cAMP) and 50 to 75 mL of Buffer E containing 1 M NaCl. The PDEactivity peak was pooled, and precipitated with ammonium sulfate (0.4g/mL enzyme pool) to remove excess cyclic nucleotide. The precipitatedproteins were resuspended in Buffer X (25 mM MOPS pH 7.5, 5 μM ZnSO₄₁ 50mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desalted via gelfiltration on a Pharmacia PD-10® column per manufacturer's instructions.The enzyme pool was dialyzed overnight against Buffer X containing 50%glycerol. This enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

[0138] The resultant preparations were about >90% pure by SDS-PAGE.These preparations had specific activities of about 10 to 50 μmol cAMPhydrolyzed per minute per milligram protein.

[0139] Preparation of PDE4C from S. cerevisiae

[0140] Yeast cells (150 g of yeast strain YI30 harboring HDUN3.48) werethawed by mixing with 100 mL glass beads (0.5 mM, acid washed) and 150mL Lysis Buffer (50 mM MOPS pH 7.2, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mMbenzamidine HCl, 5 μg/mL each of pepstatin, leupeptin, aprotinin,calpain inhibitors I and II) at room temperature. The mixture was cooledto 4° C., transferred to a BEAD-BEATER®, and the cells lysed by rapidmixing for 6 cycles of 30 sec each. The homogenate was centrifuged for22 minutes in a Beckman J2-21M centrifuge using a JA-10 rotor at 9,000rpm and 4° C. The supernatant was recovered and centrifuged in a BeckmanXL-80 ultra-centrifuge using a TI45 rotor at 36,000 rpm for 45 minutesat 4° C.

[0141] The supernatant was recovered and PDE4C was precipitated by theaddition of solid ammonium sulfate (0.26 g/mL supernatant) whilestirring in an ice bath and maintaining the pH between 7.0 and 7.5.Thirty minutes later, this mixture was centrifuged for 22 minutes in aBeckman J2 centrifuge using a JA-10 rotor at 9,000 rpm (12,000× g). Thesupernatant was discarded and the pellet was dissolved in 200 mL ofBuffer A (50 mM MOPS pH 7.5, 5 mM MgCl₂, 1 mM DTT, 2 mM benzamidine HCl,and 5 μg/mL each of leupeptin, pepstatin, and aprotinin). The pH andconductivity were corrected to 7.5 and 15-20 mS, respectively.

[0142] The resuspended sample was loaded onto a 1.6×20 cm column (25 mL)of Sigma Cibacron Blue Agarose-type 300 equilibrated in Buffer A. Thesample was cycled through the column 4 to 6 times over the course of 12hours. The column was washed in succession with 125 to 250 mL of BufferA, 125 to 250 mL of Buffer A containing 1.5 M NaCl, and then 25 to 50 mLof Buffer A. The enzyme was eluted with 50 to 75 mL of Buffer E (50 mMTris HCl pH 8, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 2 mM benzamidine HCl, and20 mM cAMP) and 50 to 75 mL of Buffer E containing 1 M NaCl. The PDE4Cactivity peak was pooled, and precipitated with ammonium sulfate (0.4g/mL enzyme pool) to remove excess cyclic nucleotide. The precipitatedproteins were resuspended in Buffer X (25 mM MOPS pH 7.2, 5 μM ZnSO₄, 50mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desalted via gelfiltration on a Pharmacia PD-10® column per manufacturer's instructions.The enzyme pool was dialyzed overnight against Buffer X containing 50%glycerol. This enzyme was quick-frozen in a dry ice/ethanol bath andstored at −70° C.

[0143] The resultant preparations were about >80% pure by SDS-PAGE.These preparations had specific activities of about 10 to 20 μmol cAMPhydrolyzed per minute per milligram protein.

[0144] Preparation of PDE4D from S. cerevisiae

[0145] Yeast cells (100 g of yeast strain YI29 harboring HDUN4.11) werethawed by mixing with 150 mL glass beads (0.5 mM, acid washed) and 150mL Lysis Buffer (50 mM MOPS pH 7.2, 10 μM ZnSO₄, 2 mM MgCl₂, 14.2 mM2-mercaptoethanol, 2 mM benzamidine HCl, 5 μg/mL each of pepstatin,leupeptin, aprotinin, calpain inhibitors I and II) at room temperature.The mixture was cooled to 4° C., transferred to a Bead-Beater®, and thecells lysed by rapid mixing for 6 cycles of 30 sec each. The homogenatewas centrifuged for 22 minutes in a Beckman J2-21M centrifuge using aJA-10 rotor at 9,000 rpm and 4° C. The supernatant was recovered andcentrifuged in a Beckman XL-80 ultracentrifuge using a TI45 rotor at36,000 rpm for 45 minutes at 4° C. The supernatant was recovered andPDE4D was precipitated by the addition of solid ammonium sulfate (0.33g/mL supernatant) while stirring in an ice bath and maintaining the pHbetween 7.0 and 7.5. Thirty minutes later, this mixture was centrifugedfor 22 minutes in a Beckman J2 centrifuge using a JA-10 rotor at 9,000rpm (12,000×9). The supernatant was discarded and the pellet wasdissolved in 100 mL of Buffer A (50 mM MOPS pH 7.5, 10 μM ZnSO₄, 5 mMMgCl₂₁ 14.2 mM 2-mercaptoethanol, 100 mM benzamidine HCl, and 5 μg/mLeach of leupeptin, pepstatin, aprotinin, calpain inhibitor I and II).The pH and conductivity were corrected to 7.5 and 15-20 mS,respectively.

[0146] At a flow rate of 0.67 mL/min, the resuspended sample was loadedonto a 1.6×20 cm column (10 mL) of Sigma Cibacron Blue Agarose-type 300equilibrated in Buffer A. The column was washed in succession with 50 to100 mL of Buffer A, 20 to 30 mL of Buffer A containing 20 mM 5¹-AMP, 50to 100 mL of Buffer A containing 1.5 M NaCl, and then 10 to 20 mL ofBuffer C (50 mM Tris HCl pH 8, 10 μM ZnSO₄, 14.2 mM 2-mercaptoethanol, 2mM benzamidine HCl). The enzyme was eluted with 20 to 30 mL of Buffer Ccontaining 20 mM cAMP.

[0147] The PDE4D activity peak was pooled and precipitated with ammoniumsulfate (0.4 g/mL enzyme pool) to remove excess cyclic nucleotide. Theprecipitated proteins were resuspended in Buffer X (25 mM MOPS pH 7.2, 5μM ZnSO₄, 50 mM NaCl, 1 mM DTT, and 1 mM benzamidine HCl) and desaltedvia gel filtration on a Pharmacia PD-10® column per manufacturer'sinstructions. The enzyme pool was dialyzed overnight against Buffer Xcontaining 50% glycerol. This enzyme preparation was quick-frozen in adry ice/ethanol bath and stored at −70° C.

[0148] The resultant preparations were about >80% pure by SDS-PAGE.These preparations had specific activities of about 20 to 50 μmol cAMPhydrolyzed per minute per milligram protein. Example No. MolecularWeight PDE4 IC₅₀ (nM) 1 445.58 4,731.1

[0149] The data presented above shows that the present compounds arepotent inhibitors of PDE4, e.g., the compounds have an IC₅₀ vs. humanrecombinant PDE4 of about 0.05 μM to about 15 μM. Preferred compoundshave an IC₅₀ of about 100 μM or less, and especially preferred compoundshave an IC₅₀ of about 50 μM or less.

[0150] The compounds of the present invention are useful for selectivelyinhibiting PDE4 activity in a mammal, without exhibiting the adverse CNSand emetic effects associated with prior PDE4 inhibitors.

[0151] Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and, therefore, only such limitations should be imposed asare indicated by the appended claims.

What is claimed is:
 1. A compound having the formula:

wherein R¹ is lower alkyl, bridged alkyl, aralkyl, cycloalkyl, a 5- or6-membered saturated heterocycle, C₁₋₃alkylenecycloalkyl, aryl- orheteroaryl-substituted propargyl, aryl- or heteroaryl-substituted allyl,or halocycloalkyl; R² is hydrogen, methyl, or halo-substituted methyl;R³ is selected from the group consisting of C(═O)OR⁷ ₁ C(═O)R⁷,C(═NH)NR⁸R⁹, C(═O)NR⁸R⁹ ₁ lower alkyl, bridged alkyl, cycloalkyl,haloalkyl, halocycloalkyl, C₁₋₃alkylenecycloalkyl, a 5- or 6-memberedsaturated heterocycle, aryl, heteroaryl, heteroarylSO₂, aralkyl,alkaryl, heteroaralkyl, heteroalkaryl, C₁₋₃ alkyleneC(═O)OR⁷,C(═O)C₁₋₃alkyleneC(═O)OR⁷, C₁₋₃alkyleneheteroaryl, C(═O)C(═O)OR⁷,C(═O)C₁₋₃alkyleneC(═O)OR⁷, C(═O)C₁₋₃alkyleneNHC(═O)OR⁷,C(═O)C₁₃alkyleneNH₂, and NH₂C(═O)OR⁷; R⁴ is hydrogen, lower alkyl,haloalkyl, cycloalkyl, aryl, C(═O)R⁷, OR⁸, NR⁸R⁹, or SR⁸; R⁵ ishydrogen, lower alkyl, haloalkyl, cycloalkyl, or aryl; R⁷ is selectedfrom the group consisting of cycloalkyl, branched or unbranched loweralkyl, heteroaryl, aryl, a heterocycle, aralkyl, and R⁷ is optionallysubstituted with one or more of RO⁸, NR⁹R⁹, or SR⁸; and R⁸ and R⁹, sameor different, are selected from the group consisting of hydrogen, loweralkyl, cycloalkyl, aryl, heteroaryl, alkaryl, heteroaralkyl,heteroalkaryl, and aralkyl, or R⁸ and R⁹ are taken together form a4-membered to 7-membered ring; R¹⁰ is hydrogen, alkyl, haloalkyl,cycloalkyl, aryl, C(═O)alkyl, C(═O)cycloalkyl, C(═O)aryl, C(═O)Oalkyl,C(═O)Ocycloalkyl, C(═O)aryl, CH₂OH, CH₂Oalkyl, CHO, CN, NO₂, or SO₂R¹¹;and R¹¹ is alkyl, cycloalkyl, trifluoromethyl, aryl, aralkyl, or NR⁸R⁹.2. The compound of claim 1 having the structure:


3. The compound of claim 1 wherein R¹ is selected from the groupconsisting of:


4. The compound of claim 1 wherein R²is methyl or difluoromethyl.
 5. Thecompound of claim 1 wherein R³ is selected from the group consisting ofC(═O)OR⁷, aralkyl, alkaryl, heteroaralkyl, heteroalkaryl, lower alkyl,cycloalkyl, heteroaryl, a heterocycle, and aryl.
 6. The composition ofclaim 1 wherein R is selected from the group consisting of:


7. The compound of claim 1 wherein R⁷ is selected from the groupconsisting of hydrogen, methyl, trifluoromethyl, cyclopropyl, acetyl,ethynyl, benzoyl, phenyl, and NR⁸R⁸, wherein R⁸ and R⁹, independently,are hydrogen, lower alkyl, or cycloalkyl, or R⁸ and R⁹ form a 5-memberedor a 6-membered ring.
 8. The compound of claim 1 wherein R⁵ is hydrogen.9. The compound of claim 1 wherein R⁷ is lower alkyl.
 10. The compoundof claim 1 wherein R⁸ and R⁹, independently, are hydrogen or loweralkyl, or are taken together form a 5-membered or 6-membered ring. 11.The compound of claim 1 wherein R is selected from the group consistingof cyclopentyl, tetrahydrofuryl, indanyl, norbornyl, phenethyl, andphenylbutyl; R² is selected from the group consisting of methyl anddifluoromethyl; R³ is selected from the group consisting of CO₂CH₃,C(═O)CH₂O H, C(═O)CH(CH₂)—OH, C(═O)C(CH₃)₂OH, and

R⁴ is NR⁸R⁹; R⁵ is hydrogen; R⁷ is methyl; R⁸ and R⁹, independently, areselected from the group consisting of hydrogen and lower alkyl, or aretaken together form a 5-membered or 6-membered ring; and R¹⁰ ishydrogen.
 12. The compound of claim 1 having a formula:


13. The compound of claim 1 having an IC₅₀ vs. human recombinant PDE4 ofabout 0.05 μM to about 15 μM.
 14. The compound of claim 1 having an IC₅₀vs. human recombinant PDE4 of about 100×10⁻⁶ M or less.
 15. The compoundof claim 1 having an IC₅₀ vs. human recombinant PDE4 of about 50×10⁻⁶ Mor less.
 16. A pharmaceutical composition comprising a compound of claim1, a pharmaceutically acceptable carrier, and, optionally, a secondanti-inflammatory therapeutic agent.
 17. The composition of claim 16wherein the second antiinflammatory therapeutic agent is capable oftargeting TNFα.
 18. A method of treating a mammal having a conditionwhere inhibition of a cAMP-specific PDE is of therapeutic benefit, saidmethod comprising administering to said mammal at therapeuticallyeffective amount of a compound of claim
 1. 19. A method of modulatingcAMP levels in a mammal comprising administering to said mammal aneffective amount of a compound of claim
 1. 20. A method of treating amammal having a condition where inhibition of a cAMP-specific PDE is ofa therapeutic benefit comprising administering to said mammal aneffective amount of a pharmaceutical composition comprising a compoundof claim 1 and a pharmaceutically acceptable carrier.
 21. The method ofclaim 20 wherein the condition is an allergic disease, an autoimmunedisease, an inflammatory disease, an arthritic disease, or dermititis.22. The method of claim 20 wherein the condition is rheumatoidarthritis, osteoarthritis, gouty arthritis, or spondylitis.
 23. Themethod of claim 20 wherein the condition is thyroid-associatedophthalmopathy, Behcet disease, sepsis, septic shock, endotoxic shock,gram negative sepsis, gram positive sepsis, toxic shock syndrome,allergic conjunctivitis, vernal conjunctivitis, or eosinophilicgranuloma.
 24. The method of claim 20 wherein the condition is asthma,chronic bronchitis, allergic rhinitis, adult respiratory distresssyndrome, chronic pulmonary inflammatory disease, chronic obstructivepulmonary disease, silicosis, or pulmonary sarcoidosis.
 25. The methodof claim 20 wherein the condition is reperfusion injury of themyocardium, brain or extremities as a brain or spinal cord injury due totrauma.
 26. The method of claim 20 wherein the condition is a fibrosis,keloid formation, or scar tissue formation.
 27. The method of claim 20wherein the condition is systemic lupus erythematosus, a transplantrejection disorder, a graft vs. host reaction, or an allograftrejection.
 28. The method of claim 20 wherein the condition is chronicglomerulonephritis, an inflammatory bowel disease, Crohn's disease, orulcerative colitis.
 29. The method of claim 20 wherein the condition isproliferative lymphocytic disease or a leukemia.
 30. The method of claim20 wherein the condition is an inflammatory dermatosis, atopicdermatitis, psoriasis, or urticaria.
 31. The method of claim 20 whereinthe condition is a cardiomyopathy, congestive heart failure,atherosclerosis, pyrexia, cachexia, cachexia secondary to infection ormalignancy, cachexia secondary to acquired immune deficiency syndrome,ARC, cerebral malaria, osteoporosis, a bone resorption disease, feverand myalgias due to infection, erectile dysfunction, diabetes insipidus,a central nervous system disorder, depression, or multi-infarctdementia, an anxiety or stress response, cerebral ischemia, tardivedyskinesia, Parkinson's disease, or premenstrual syndrome.
 32. Themethod of claim 20 wherein the mammal exhibits a minimal emeticresponse.
 33. The method of claim 20 wherein the mammal is free of anemetic response.
 34. The method of claim 20 wherein the mammal exhibitsminimal adverse central nervous system side effects.
 35. The method ofclaim 20 wherein the mammal is free of adverse central nervous systemside effects.
 36. The method of reducing TNF levels in a mammalcomprising administering to said mammal therapeutically effective amountof a compound of claim
 1. 37. A method of suppressing inflammatory cellactivation in a mammal comprising administering to said mammal atherapeutically effective amount of a compound of claim
 1. 38. A methodof inhibiting PDE4 function in a mammal comprising administering to saidmammal a therapeutically effective amount of a compound of claim 1.